Dynamic Catalysis Guided by Nucleic Acid Networks and DNA Nanostructures

Nucleic acid networks conjugated to native enzymes and supramolecular DNA nanostructures modified with enzymes or DNAzymes act as functional reaction modules for guiding dynamic catalytic transformations. These systems are exemplified with the assembly of constitutional dynamic networks (CDNs) composed of nucleic acid-functionalized enzymes, as constituents, undergoing triggered structural reconfiguration, leading to dynamically switched biocatalytic cascades. By coupling two nucleic acid/enzyme networks, the intercommunicated feedback-driven dynamic biocatalytic operation of the system is demonstrated. In addition, the tailoring of a nucleic acid/enzyme reaction network driving a dissipative, transient, biocatalytic cascade is introduced as a model system for out-of-equilibrium dynamically modulated biocatalytic transformation in nature. Also, supramolecular nucleic acid machines or DNA nanostructures, modified with DNAzyme or enzyme constituents, act as functional reaction modules driving temporal dynamic catalysis. The design of dynamic supramolecular machines is exemplified with the introduction of an interlocked two-ring catenane device that is dynamically reversibly switched between two states operating two different DNAzymes, and with the tailoring of a DNA-tweezers device functionalized with enzyme/DNAzyme constituents that guides the dynamic ON/OFF operation of a biocatalytic cascade by opening and closing the molecular device. In addition, DNA origami nanostructures provide functional scaffolds for the programmed positioning of enzymes or DNAzyme for the switchable operation of catalytic transformations. This is introduced by the tailored functionalization of the edges of origami tiles with nucleic acids guiding the switchable formation of DNAzyme catalysts through the dimerization/separation of the tiles. In addition, the programmed deposition of two-enzyme/cofactor constituents on the origami raft allowed the dynamic photochemical activation of the cofactor-mediated biocatalytic cascade on the spatially biocatalytic assembly on the scaffold. Furthermore, photoinduced “mechanical” switchable and reversible unlocking and closing of nanoholes in the origami frameworks allow the “ON” and “OFF” operation of DNAzyme units in the nanoholes, confined environments. The future challenges and potential applications of dynamic nucleic acid/enzyme and DNAzyme conjugates are discussed in the conclusion paragraph.


INTRODUCTION
The information encoded in the base sequence of DNA includes substantial structural and functional information. This includes instructive structural reversible reconfiguration properties, such as dictated strand displacement of duplex structures, 1,2 the reconfiguration of guanosine-rich strands, in the presence of K + -ions, into G-quadruplexes and their separation by crown-ethers, 3 the pH-induced formation and separation of i-motif cytosine-rich sequences, 4 the formation and dissociation of triplex nucleic acid structures, 5 and the light-stimulated stabilization and dissociation of nucleic acid duplexes, by photoisomerizable intercalators, such as trans/cis azobenzene. 6,7 Functional information dictated by the base sequence of nucleic acids includes sequence-specific recognition properties of low-molecular-weight substrates or macromolecules (aptamers) 8,9 or sequence-guided catalytic functions of the nucleic acids, e.g., hemin/G-quadruplex DNAzymes 10,11 or cofactor-dependent DNAzymes. 12,13 The reversible reconfiguration of nucleic acids by auxiliary triggers was applied to develop diverse DNA switches 14 and DNA machines, 15−17 such as tweezers, 18,19 walkers, 20−22 interlocked catenated rings, 23,24 and more. 25,26 The recognition and catalytic functions of nucleic acids were broadly applied for the development of sensing platforms, 27−29 amplifying agents for electrochemical or optical sensors, 30−32 engineering of catalytic and photocatalytic supramolecular assemblies, 33−36 and their therapeutic applications, including targeting of cells 37−39 or inhibition of proteins, 40 and more. In addition, the coded basesequence of nucleic acids was extensively used to develop programmed linear structures and to assemble 2D 41,42 and 3D nanostructures, such as DNA origami 41,43 or DNA tetrahedra. 44 Nucleic acid−protein conjugates or DNA−aptamer conjugates were engineered in spatially proximate positions on the DNA scaffolds to emulate biocatalytic cascades in confined native environments. 45 Indeed, efficient bicatalytic cascades, as compared to an analog biocatalytically coupled reaction in nonorganized, random mixture of enzymes, were demonstrated. 46,47 Furthermore, intracellular dynamic interactions between DNA, RNA, and proteins form complex biological networks triggered by environmental physical or chemical stimuli and operate under thermodynamic control or under transient, outof-equilibrium, dissipative conditions. The intercommunication between these dynamic networks leads to programmed reaction patterns involving adaption, 48,49 amplification, 50 feedback 51 or oscillatory 52 mechanisms, signal propagation, 53 switching, 54,55 temporal and multistable behaviors, 56 and clustering 48 of networks into gated or cascaded assemblies. Diverse complex bioprocesses are driven by these networks, including cell differentiation, 48 regulation of gene transcription, 57 morphogenesis, 58 and eventually to genome instability and stress-induced oncogene replication and cancer. 59−61 In vitro design of chemical circuits mimicking natural dynamic networks attracts broad interest within the general topic of Systems Chemistry. 62−64 The structural and functional properties of nucleic acids have been utilized to assemble dynamic DNA networks and reaction moduli triggered by different auxiliary triggers. These include the supramolecular engineering of constitutional dynamic DNA networks 65 that demonstrated reversible adaptive, 66,67 hierarchically adaptive, 68 feedback-driven, 69 and intercommunicating functionalities 70 in the presence of auxiliary triggers. Also, transient, out-of-equilibrium, dissipative signal-triggered DNAbased frameworks were designed, including transcriptional oscillators 71 or bistable regulatory networks 72 and DNA networks coupled to biocatalysts, e.g., polymerase/endonuclease/nickase leading to oscillatory behavior. 73−75 Also, fueltriggered transient enzyme-coupled (nickase) networks, 76 fueltriggered transient DNAzyme systems, 77 and light-or aptamerguided transient ligation cycles 78,79 were accomplished. Different applications of dynamically reconfigured DNA assemblies were introduced including the use of constitutional dynamic networks for controlling hydrogel stiffness for controlled drug release 80 and for the dynamic network-guided release of drugs from nanocarriers. 81 Transient DNA networks were applied as functional frameworks for the temporal uptake and release of loads, 82 the photoacid-driven dissipative polymerization/depolymerization of DNA fibers, 83 and the temporal aggregation/deaggregation of Au nanoparticles or CdSe/ZnS semiconductor quantum dots and the control over their optical properties. 84 To emulate functions of native systems, it is important, however, to couple the trigged dynamic reconfiguration of DNA networks to dynamic biocatalytic processes. This can be accomplished by the conjugation of enzymes to dynamically triggered networks or by tethering biocatalysts to a dynamically switchable DNA framework. This Review discusses switchable catalytic properties and the control over biocatalytic cascades, by means of dynamic DNA networks conjugated to enzymes or by enzymes/DNAzymes conjugated to switchable DNA nanostructures.
2.1. Controlling Biocatalytic Cascades by Nucleic Acid-Based Constitutional Dynamic Networks (CDNs). Figure 1A depicts schematically the concept of a 2 × 2 constitutional dynamic network (CDN) composed of four constituents, AA′, BB′, AB′, and BA′. The four constituents exist in an equilibrated state X, where the contents of the constituents are controlled by their relative stabilities. 85 Subjecting CDN X to an auxiliary trigger T 1 that stabilizes, for example, constituent AA′ reconfigures CDN X into CDN Y where constituent AA′ is upregulated (increased in its equilibrated content) at the expense of constituents AB′ and BA′ that are downregulated and the concomitant increase in the content of BB′ (as a result of the recombination of B and B′ being separated upon supplying A and A′ to enrich AA′). Applying the counter trigger T 1 ′ that destabilizes the constituent AA′ associated with CDN Y by displacement of T 1 regenerates the equilibrated CDN X. Similarly, applying the trigger T 2 that stabilizes constituent AB′ of CDN X reconfigures CDN X into CDN Z where AB′ is upregulated; the constituents AA′ and BB′, sharing components with AB′, are downregulated, and concomitantly, the constituent BA′ is upregulated. In addition, subjecting CDN Z to the trigger T 2 ′, Figure 1. (A) Schematic assembly of a constitutional dynamic network (CDN) composed of four equilibrated constituents, CDN X, being dynamically reconfigured by auxiliary triggers. The constituents are composed of four components A, A′, B, B′, yielding the equilibrated constituents AA′, BB′, AB′, and BA′. The T 1 -triggered stabilization of constituent AA′ in CDN X leads to the dynamic reconfiguration of CDN X into the re-equilibrated CDN Y where AA′ is enriched at the expense of AB′ and BA′, resulting the concomitant enrichment of BB′ in the equilibrated mixture. Subjecting CDN X to trigger T 2 stabilizes the constituent AB′ and as a result leads to the dynamic reconfiguration of CDN X to Z, where constituents AB′ and BA′ are enriched and constituents AA′ and BB′ are downregulated in their content in the resulting and BB′ re-equilibrated network. Adapted from ref 65 with permission. Copyright 2020, American Chemical Society. (B) Examples of reversible triggers that guide the programmed dynamic reconfiguration of CDNs. that destabilizes AB′, results in the recovery of the regenerated CDN X.
The information encoded in nucleic acids provides a versatile means to construct constituents of pre-engineered stabilities as scaffolds comprising the CDN. In addition, the nucleic acid constituents include in their scaffolds triggerable units that allow the reversible stabilization/destabilization of their structures ( Figure 1B). These units might include a K +ion G-quadruplex/crown ether switchable unit, 3 a triplex stabilizing stand being displaced by a counter strand, 5 a photoismerizable trans/cis azobenzene duplex intercalator stabilizing/destabilizing unit, 6,7 or metal-ion bridged stabilized duplex, e.g., T-Hg 2+ -T or C-Ag + -C, being separated by a ligand such as cysteine. 86−88 In addition, the constituent should be coupled to a functional element that quantitatively transduces content of the constituent upon the dynamic reconfiguration of the network. For example, by the integration of a catalytic nucleic acid into the structures of the constituents, e.g., Mg 2+ion-dependent DNAzymes, the rates of the DNAzymecatalyzed cleavage of fluorophore-quencher modified substrates, and the use of appropriate calibration curves, provide a means to quantitatively report the contents of the constituents in the dynamic systems. Indeed, substantial progress was recently accomplished in designing and operating CDNs. 65 Different triggers such as G-quadruplex formation and dissociation by K + -ions/crown ether, 67 the formation and (B) Synthesis of enzymes functionalized with single nucleic acid tether A-GOx and A′-HRP. (C) Contents of the equilibrated constituents in CDN "L", CDN "M", and CDN "N", evaluated by the activities of the Mg 2+ -ion-dependent DNAzymes associated with the constituents. (D) Time-dependent absorbance changes of ABTS •-, reflecting the efficacy of the GOx/HRP biocatalytic cascade associated with CDN "L", panel I, curve (i), and T 1 -triggered reconfigured CDN "M", panel I, curve (ii); Time-dependent absorbance changes of ABTS •reflecting the efficacy of the GOx/HRP biocatalytic cascade associated with CDN "L", panel II, curve (i), and T 2 -triggered reconfigured CDN "N", panel II, curve (ii). (E) Panel I-Cyclic operation of the GOx/HRP biocatalytic cascade upon the reversible switching, between CDN "L" and CDN "M" using the triggers T 1 and T 1 ′, and panel II-Cyclic operation of the GOx/HRP biocatalytic cascade upon the reversible switching, between CDN "L" and CDN "N" using the triggers T 2 and T 2 ′. Bioconjugate Chemistry pubs.acs.org/bc Review displacement of triplex nucleic acids, 89 and the photoisomerization of azobenzene-modified intercalating strands 90 were applied to reconfigure CDNs. Adaptive and hierarchically adaptive functions of dynamic networks, 68 intercommunication 91 and feedback-driven networks, 69 and engineering of higher-order networks, such as three-dimensional networks, 92 were demonstrated. Different applications of dynamic networks were introduced including the use of CDNs as functional units to stimulate dictated drug release 81 or guided stiffness properties of hydrogel matrices and their use for switchable drug release and self-healing materials. 80 Here we exemplify the conjugation of biocatalytic units to CDNs and the application of the hybrid matrices to stimulate dynamically controlled biocatalytic cascades and intercommunicated biocatalytic cascades. The operation of biocatalytic cascades in confined microenvironments of spatially proximate positions has been a subject of extensive experimental and theoretical research as a means to emulate biocatalytic processes in confined cellular media. 93,94 The steric proximity between communicating catalysts where the product of one biocatalyst acts as the substrate of the neighboring biocatalyst proved to be an effective means to enhance biocatalytic cascades. Indeed, proximity effects induced by intercommunicated enzyme-loaded microdroplets, 95 spatial positioning of biocatalysts on DNA scaffolds 96 or origami tiles, 97 or tethering enzymes on peptide or receptor frameworks 98,99 proved a versatile means to spatially control biocatalytic cascades. In addition, the covalent tethering of biocatalysts on dynamically triggered DNA frameworks, e.g., tweezers, enabled the dynamic spatial control over the effectiveness of biocatalytic cascades. 100 The covalent tethering of biocatalysts to constituents of CDNs allows not only the spatial control of proximity of intercommunicating biocatalysts but also the dynamic control over the content of the intercommunicating biocatalysts by means of the trigger reconfiguration of the DNA network. Figure 2A depicts the assembly of a DNA-based constitutional dynamic network, CDN L, that guides the dynamic spatial control over the intercommunicated cascade between glucose oxidase, GOx, and horseradish peroxidase, HRP. 101 GOx is functionalized by nucleic acid component A, whereas the HRP is modified with the component A′. As a result, CDN Figure 3. Orthogonal coupled operation of two constitutional dynamic networks that guide the dynamic cascaded biocatalytic reactions consisting of GOx/HRP and ADH/NAD + . The two CDNs "L" and "P" are subjected to two hairpins H aa ′ and H dd ′ that fuel the coupled operation of the networks. Cleavage of H aa ′ by constituent CC′ associated with CDN "P" yields fragment H aa-1 that stabilizes constituent AA′ in CDN L resulting in the temporal dynamic enrichment of AA′ and the concomitant enrichment of constituent BB′. Simultaneously, cleavage of hairpin H dd ′ by constituent BB′ associated with CDN L yields strand H dd-1 ′ that stabilized constituent DD′ of CDN P and the concomitant dynamic enrichment of constituent CC′. The operation of the two networks leads to the dynamic temporal enrichment of AA′ associated with CDN L and to the accompanying temporal feedback-driven enhancement of the GOx/HRP cascade ( Figure 3A (Figure 2A, panel II). The chemistry involved in the modification of the enzymes with a single nucleic acid is depicted in Figure 2B.    Figure 2C depicts the contents (concentrations) of the constituents in CDN L, the T 1 -triggered CDN M, and the T 2 -triggered, reconfigured, CDN N, reported by the activities of the Mg 2+ -ion-dependent DNAzymes coupled to the respective constituents. The contents of constituents AA′ and BB′ in CDN M increase by 49% and 53% and of AB′ and BA′ decrease by 58% and 76% as compared to the contents of their constituents in CDN L. Similarly, the reconfiguration of CDN L into CDN N is accompanied by the decrease of the contents of AA′ and BB′ by 35% and 48% and the increase in the concentrations of AB′ and BA′ by 54% and 100%, respectively. The triggered control over the concentrations of constituent AA′ in the three network controls the effectiveness of the GOx/HRP bicatalytic cascade ( Figure 2D). The T 1triggered transition of CDN L to CDN M is accompanied by a 2.1-fold enhancement of the biocatalytic cascade, panel I, whereas the T 2 -triggered transformation of CDN L to CDN N results in a 0.6-fold inhibition of the biocatalytic cascade, panel II. By the reversible transition of CDN L to CDN M and CDN N by triggering T 1 /T 1 ′ and T 2 /T 2 ′, the activities of the respective biocatalytic cascade are cycled, and these follow the CDN-guided control over the concentrations of the enzymes (GOx and HRP) participating in the spatially intimate configuration that activates the bicatalytic cascades ( Figure  2E, panel I and panel II). In analogy, a constitutional dynamic network driving the alcohol dehydrogenase, ADH, catalyzed the reduction of NAD + to NADH, and the subsequent NADH mediated reduction of pyruvic acid to lactic acid, in the presence of lactate dehydrogenase, LDH, was achieved. 101 The triggered reconfiguration of the CDN between upregulated and downregulated equilibrated configurations of the biocatalytic constituents was exploited, demonstrating the versatility of CDNs to control biocatalytic cascades. Most importantly, however, the two CDNs-guided biocatalytic cascades comprising GOx/HRP and ADH/NAD + were coupled to yield the CDNs-driven dynamic feedback operation of the two cascades ( Figure 3A). Two hairpins, H aa ′ and H dd ′, were introduced into the mixture of CDN P and CDN L. Constituent CC′ associated with CDN P cleaves H aa′ to yield the fragmented strand H aa′-1 that stabilized constituent AA′ of CDN L, while constituent BB′ associated with CDN L cleaves hairpin H dd′ to yield the fragmented product H dd′-1 that stabilizes constituent DD′ of CDN P. As a result, the intercommunication of the two networks leads to dynamic temporal feedback-driven reconfiguration of the two CDNs, where the biocatalytic cascade GOx/HRP associated with the constituent AA′, being part of CDN L, reveals a temporal feedback-driven increase in its effectiveness of the GOx/HRP cascade (time-dependent increase in the catalytic rate of generated ABTS •-) ( Figure 3B), and a feedback-driven increase in the content of constituent DD′ in CDN P and an enhancement in the effectiveness of the ADH/NAD + cascade (time-dependent increase in the catalytic rate of generating NADH, monitored by the NADH-mediated reduction of methylene blue, MB + to MBH) ( Figure 3C).
The dynamic control of chemical reactions by means of CDNs was extended to include CDN-guided photocatalytic processes, and particularly artificial photosynthetic cascades. 102 Figure 4A outlines the photoactive constitutional dynamic network acting as the artificial photosynthetic reaction module. The network, CDN Z, includes four constituents AA′, BB′, AB′, and BA′, where the components A and A′ composing constituent AA′ are modified with the Zn(II)-protoporphyrin IX, Zn(II)-PPIX, loaded photosensitizer, and the N,N′-dialkyl-4,4′-bipyridinium, V 2+ , electron acceptor. Each of the constituent in CDN Z includes a biloop region that can be stabilized by an auxiliary strand, T 1 , through the formation of a T-A•T triplex. In addition, each of the constituents is functionalized with a different Mg 2+ -ion-dependent DNAzyme unit that provides a catalytic label that reports the contents (concentrations) of the respective constituents via the DNAzyme catalyzed cleavage of the respective fluorophore/ quencher-modified substrates and the use of appropriate calibration curves. Subjecting CDN Z to trigger T 1 stabilizes constituent AA′, by forming a T-A•T triplex structure in the biloop domain, resulting in the reconfiguration of CDN Z to CDN Z a where AA′ is upregulated, AB′ and BA′ are downregulated, and concomitantly BB′ is upregulated. Treatment of CDN Z a with the counter trigger T 1 ′ separates the triplex structure, thereby restoring CDN Z. Similarly, subjecting CDN Z to trigger T 2 stabilizes BA′ and reconfigures CDN Z to CDN Z b where constituents BA′ and AB′ are upregulated and the contents of constituents AA′ and BB′ are downregulated. Subjecting CDN Z b to the counter trigger T 2 ′ restores the parent CDN Z. The concentrations of the constituents in the three CDNs are transduced by the Mg 2+ion-dependent DNAzymes reporter units and displayed in Figure 4B. The functionalization of constituent AA′ in the different CDNs with the Zn(II)-PPIX photosensitizer and bipyridinium (V 2+ ) electron acceptor units leads to the photoinduced electron transfer within the photosensitizer/ electron acceptor pair resulting in Zn(II)-PPIX +• and V +• and the subsequent oxidation of Zn(II)-PPIX +• by sacrificial electron donor (RSH) to the disulfide (RSSR) product ( Figure 4A, panel I). The resulting photogenerated bipyridinium radical cation mediates in the ferredoxin NADP reductase, FNR, catalyzed reduction of NADP + to NADPH. The dynamic control over the content of the photoactive photosensitizer/electron acceptor diad in the respective CDNs controls the effectiveness of the biocatalyzed generated NADPH and the switchable reconfigurations of the CDNs lead to switchable photobiocatalytic transformations. Figure  4C shows the absorbance spectra of the V +• generated in the CDNs Z, Z a , and Z b under steady-state illumination of the systems. The concentration of generated V +• is dictated by the contentment of constituent AA′. Figure 4D depicts the switchable generation of CDNs-dictated V +• by the different reconfigured CDNs. Figure 4E  Alternatively, the biocatalytic assembly T 1 /A 1 -GOx + A 2 -HRP activates the biocatalytic cascade where the aerobic oxidation of glucose leads to the coupled HRP-catalyzed generation of chemiluminescence through the oxidation of luminol ( Figure  5A, inset Y). The duplex L 1 ′/L 1 was engineered, however, to be nicked by Nt.BbvCI, and the cleaved strand L 1 ′ yielded fragmented "waste" products that were separated from L 1 . The    26 or swinging rings (catenanes or rotaxane). 23,105,106 Different stimuli, such as fuel/antifuel strand displacement, pH, metal-ion/ligand, or light, have been widely applied as triggers to dynamically modulate the mechanical reconfiguration of DNA nanostructures. In addition, the base sequences of nucleic acids provide instructive information guiding the precise positioning of nucleic acid on DNA scaffolds through dictated hybridization patterns. In particular, the versatile conjugation chemistries allowing the covalent coupling of lowmolecular-weight ligands or macromolecules (e.g., proteins) to nucleic acids provide general means to precisely position spatially programmed assemblies of molecular or macromolecular systems on nucleic acid scaffolds. That is, the hybridization of nucleic acid−protein conjugates with preengineered protruding strands associated with DNA scaffolds yields programmed, spatially ordered arrays on the DNA scaffolds. Furthermore, besides the organization of functional assemblies on one-dimensional DNA scaffolds, the programmability of nucleic acid structures allows the assembly of twodimensional nanostructures, such as Y-shaped, 107 crossover tetra-armed shaped structures 108 or self-assembled complex two-dimensional origami structures, 109 and even the functionalization of three-dimensional DNA structures, such as DNA tetrahedra, DNA origami "boxes", 110 or capsules. 111 These diverse structures enable the dictated spatial positioning of DNA-modified functionalities not only on the surfaces of the nanostructures but also at geometrically engineered edge positions of the supports. The integration of mechanically switchable properties into the DNA frameworks that are modified with spatially engineered catalytic sites is then anticipated to yield dynamic mechanically switchable catalytic assemblies and to intercommunicated switchable catalytic cascades in spatially confined environments. This section will dynamically exemplify such reconfigurable catalytic devices. Figure 6A exemplifies a supramolecular interlocked two-ring catenane DNA device for the switchable dynamic operation of a hemin/G-quadruplex catalyst. 112 The interlocked structure consists of two rings α1 and β1 where ring α1 includes sequence II hybridized with sequence II′ that is a part of ring β1. In addition, ring α1 includes sequence III that is composed of two subsequences z and w, that are blocked, through hybridization, with an auxiliary strand L 1 . The sequence I is composed of the G-rich sequence that is caged in the catenated structure, and domain II′ of ring β1 includes two subsequences p and q that are complementary to the sequence w caged in the duplex III/L 1 structure. The displacement of strand L 1 by a fuel strand L 1 ′ yields the duplex L 1 /L 1 ′, resulting in the "bare" sequence w that allows the dynamic transition of ring β1 to domain w yielding the energetically stabilized duplex between domains p/q and w. The transition of ring β1 to site w associated with ring α releases the G-rich sequence I associated with ring α1, resulting in switched-on catalyzed oxidation of ABTS 2− by H 2 O 2 to the colored product ABTS •-, in the presence of K + -ions, hemin, and the reconfigured sequence I into the hemin/G-quadruplex DNAzyme, state B. Subjecting state B to the trigger L 1 displaces ring β1 from the sequence w, the L 1 -locked sequence z/w, and the release of ring β1 that dynamically restores state A, where domain II′ in ring β1 hybridizes to domain II associated with ring α1. This leads to dissociation of the G-quadruplex structure and to switching off the catalytic activity of the DNAzyme. By the cyclic activation of the catenated device with triggers L 1 ′/L 1 , the catalytic functions of the device were dynamically switched between "ON" and "OFF" states, that were followed by the temporal formation and blockage of the colored ABTS •products ( Figure 6B and C). In a related system, a two-ring catenane system was designed to switch catalytic activities between two DNAzymes ( Figure 6D). In state X, the Mg 2+ -ion-dependent DNAzyme consisting of two catenated rings α2 and β2 is activated, and it cleaves the fluorophore F 2 (FAM)-quencher Q 2 (Iowa black FQ)-modified substrate S 2 to yield the fluorescent readout signal. In state X, the hybridization of domains III and III′ of rings α2 and β2 blocks a part of the Zn 2+ -ion-dependent DNAzyme sequence encoded in the rim of ring α2. Treatment of the two-ring catenane in state X with the fuel strand L 3 results in the displacement of ring β2 and its hybridization to the domains (g+h) of α2, while unlocking domain III is associated with ring α2. The released ring α2 forms an energetically stabilized interlocked hybrid duplex III′/ (g+h) catenated structure, and concomitantly the released sequence II reorganizes into the Zn 2+ -ion-dependent DNAzyme, state Y, that cleaves the F 1 (ROX)/quencher (Q 1 = BHQ2)-modified substrate S 1 to yield the fluorescent F 1fragment. Subjecting state Y to the counter fuel strand, L 3 ′, reconfigures state Y into state X. That is, by the cyclic treatment of the α2/β2 two-ring interlocked catenane, the switchable dynamic transitions between state X and state Y lead to the dynamic, orthogonally operating, DNAzymes (the Mg 2+ -ion-dependent DNAzyme and the Zn 2+ -ion-dependent DNAzyme) ( Figure 6E).
A three-catalyst cascade 113 operating by a dynamic tweezer device is exemplified in Figure 7A. The oligonucleotide− protein conjugates (1)-functionalized β-galactosidase, (1)-β-Gal, and (2)-modified GOx, (2)-GOx, are cross-linked by a fluorophore (F = Cy5.5)/quencher (Q = Iowa Black RQ) modified strand (4) to yield a supramolecular three-catalyst nanostructure. In addition, the two nucleic acid conjugates consisting of (1)-β-Gal and (2)-GOx were bridged by a nucleic acid (3) that self-assembles into a hemin/G-quadruplex DNAzyme cross-linking unit, in the presence of K + -ions and hemin, state G. The bifunctional cross-linked structure in state G activates the three-catalyst cascade where β-Gal hydrolyzes lactose to galactose and glucose. The resulting glucose is aerobically oxidized to gluconic acid and H 2 O 2 by GOx, and the formed H 2 O 2 provides the oxidant for the hemin/Gquadruplex-catalyzed oxidation of ABTS 2− to the colored ABTS •that provides the transduction signal for the threecatalyst cascade. Treatment of state G with 18-crown-6-ether separates the bridging hemin/G-quadruplex structure, resulting in an extended opened configuration of the tweezers, state H, in which the three-catalyst cascade is blocked. Readdition of K + -ions to the system restores the closed hemin/Gquadruplex-driven tweezers in which the three-catalyst cascade is reactivated. By the cyclic addition of 18-crown-6-ether and K + -ions, the dynamic switching of the three-catalyst cascade between ON−OFF states was demonstrated ( Figure 7B). The dynamic transitions of the tweezers device between closed and open configurations were followed by probing the fluorescence responses of the fluorophore/quencher-functionalized bridging unit ( Figure 7C). While the fluorophore is effectively quenched in the closed configuration, state G, the fluorescence of the probe is intensified in the open configuration, state H, Bioconjugate Chemistry pubs.acs.org/bc Review where spatial separation between the fluorophore and quencher exists. It should be noted, however, that although the hemin/G-quadruplex is a useful switchable DNAzyme probe in operating biocatalytic cascades, its long-term participation in cyclic cascade is limited due to its reconfiguration into different G-quadruplex motifs, revealing alternate catalytic activities. 114 Related studies have applied dynamic tweezers structures to operate switchable biocatalytic cascades. 115−117 For example, 117 a double crossover (DX) rigid structure consisting of two arms, modified at each of their ends with GOx and horseradish peroxidase (HRP), bridged by an immobile joint to yield the open tweezers structure. By the reverse displacement of the fuel strand with an antifuel hairpin, the rigid joint double crossover structure was restored. As in the condensed rigid structure, where the two enzymes GOx and HRP are in intimate contact, the bienzyme cascade whereby the aerobic GOx catalyzed oxidation of glucose to gluconic acid and H 2 O 2 proceeds, followed by the effective channeling of H 2 O 2 to the neighboring HRP that catalyzed the H 2 O 2 oxidation of ABTS 2− to the colored product ABTS •-( Figure 7D). The fueled opening of the closed structure into the open tweezers state spatially separate the two enzymes perturbing the bienzyme cascade. By the reversibly opening and closing of the tweezers structure the biocatalytic cascade was switched between low and high activity ( Figure 7E). The similar concept was adapted to apply the dynamic opening and closing to switch the glucose-6-phosphate dehydrogenase/ NAD + biocatalytic cascade. 116 Furthermore, the dynamic switchable control of enzymes was demonstrated by designing nucleic acid programmed enzyme−inhibitor scaffolds where the intimate inhibition of the enzyme is stimulated by strand displacement or light-induced control over the distance separating the enzyme/inhibitor constituents. 118−120 2.4. Dynamic Switchable Biocatalysis on DNA Frameworks. The spatial positioning of catalytic units on twodimensional DNA frameworks provides a further means to dynamically control catalytic functions of biomolecular conjugates in organized nanoenvironments and to guide programmed biocatalytically driven cascades. DNA origami scaffolds provide a remarkable structure to precisely position and dynamically activate catalytic assemblies and particularly switchable catalytic systems. The assembly of origami rafts or tiles by a programmed set of "stapler" units that links together along circular DNA enables the integration of functional (catalytic) nucleic acids at the edges of the origami tiles or the functionalization of predesigned positions of the upper or lower origami surfaces by the extension of stapler units with protruding tethers to which nucleic acid-modified catalyst are anchored. Figure 8A exemplifies the switchable dynamic separation and formation of an origami dimer structure that leads to the reversible assembly of hemin/G-quadruplex units at the edge of the origami-tiles. 121 The origami dimer consists of two tiles A and B interbridged by complementary nucleic acids L 1 /L 2 and L 3 /L 4 integrated at the edges of the origami tiles A and B (marked with two "dots" composed of 3 × surface-tethered hairpins). The nucleic acids L 2 and L 3 include guanosine-rich tethers g 1 partially caged in the duplex a/a′ and b/b′ domains bridging the duplexes L 1 /L 2 and L 3 /L 4 . Treatment of the AB dimers with K + -ions separates the dimers while self-assembling the tethers L 2 , L 4 into K + -stabilized Gquadruplexes linked to the edge of tile B. In the presence of hemin, the resulting hemin/G-quadruplex units provide catalytic units for the H 2 O 2 -catalyzed oxidation of Amplex Red to the fluorescent Resorufin ( Figure 8A, inset). Subjecting the separated origami tiles to 18-crown-6-ether dissociates the hemin/G-quadruplex units leading to the energetically stabilized catalytically inactive AB dimers. The switchable separation and reformation of the origami dimers AB was followed by atomic force microscopy (AFM) (Figure 8B), and the dynamic switchable catalytic functions of the hemin/Gquadruplex catalyzed oxidation of Amplex Red were demonstrated ( Figure 8C).

Bioconjugate Chemistry pubs.acs.org/bc Review
The dynamic light-induced switchable NAD + mediated activation of the biocatalytic cascade between glucose-6phophate dehydrogenase (G6pDH) and lactate dehydrogenase (LDH) on a two-dimension origami tile is displayed in Figure  9A using a phototriggered NAD + -mediated swinging arm. 122 The enzyme conjugates G6pDH and LDH modified with nucleic acid tethers were positioned in spatially proximate sites on the origami tile. The NAD + cofactor was linked through a Holliday Junction structure (23), positioned in between the two enzymes on the tile framework. The Holliday Junction unit includes a swinging arm (24) that forms a stable duplex with a trans-azobenzene foothold tether (25). Formation of the trans-azobenzene functionalized (24)/(25) duplex swings the NAD + -cofactor away from the two enzyme components prohibiting the cofactor-induced communication between the biocatalysts. Photoisomerization of the trans-azobenzene units to the cis-states separates the duplex (24)/(25) allowing the "mechanical" swinging of the NAD + -cofactor unit into spatial proximity to the two biocatalysts. This switches on the G6pDH catalyzed oxidation of glucose-6-phosphate to D-glucono-1,5lactone 6-phosphate with the concomitant reduction of NAD + to NADH (eq 1). The resulting reduced cofactor mediates the reduction of pyruvic acid to lactic acid (eq 2).
Thus, the light-induced swinging of the cofactor into the proximity of the two enzymes switches on the biocatalytic cascade. The reverse photoisomerization of the cis-azobenzene units to the trans-states swings back the cofactor arm to the inactive state of the origami device. By the cyclic photoisomerization of the azobenzene units between cis and trans states, the dynamic swinging of the NAD + cofactor arm enabled the "ON" and "OFF" operation of the biocatalytic cascade ( Figure 9B).
The dynamic rotational control over a biocatalytic cascade operating on an origami scaffold consisting of a hexagonal frame was demonstrated 123 (Figure 10). The six interedges of the frame were functionalized with six hairpin tethers (i)−(vi). The enzyme HRP is firmly positioned on the hexagonal DNA origami surface through hybridization to a protruding tether. A three-arm stator comprising nucleic acid I-linked to the enzyme GOx and nucleic acids II and III was positioned in the void volume of the origami frame by linking the tethers I to (i), II to (iii), and III to (v) through bridging nucleic acids L 1 , L 2 , and L 3 , resulting in state P 1 ( Figure 10A). Subjecting the device in state P 1 to the displacement strand L 1 ′, L 2 ′, and L 3 ' and to bridging strand L 1 a , L 2 a , and L 3 a results in the rotation of the stator to state P 2 , where the stator arms are fixed to tethers (ii), (iv), and (vi) using the bridges L 1 a , L 2 a , and L 3 a . The subsequent treatment of state P 2 with the counter strands L 1 a ′, L 2 a ′, and L 3 a ′ and the helper strands, L 1 b , L 2 b , and L 3 b result in a further rotation step to yield state P 3 . These dynamic rotations lead to the control over the spatial separation of the enzymes, GOx and HRP. While in state P 1 the two biocatalysts are in proximity, in state P 2 the two biocatalysts are partially spatially separated, and in state P 3 the biocatalysts are spatially apart. As a result, the biocatalytic cascade, consisting of the GOx-mediated aerobic oxidation of glucose to gluconic acid and H 2 O 2 and the subsquent HRP catalyzed oxidation of ABTS 2− to ABTS •by H 2 O 2 is most effective in state P 1 , and it gradually decreases to lower activity in state P 2 and lowest activity in state P 3 ( Figure 10B). This is, indeed, reflected by the order of the rates of the biocatalytic cascade generating ABTS •by the respective states P 1 , P 2 , and P 3 ( Figure 10C).
The dynamic "mechanical" light-induced opening and closing of a nanohole in a DNA origami tile, and the switchable operation of a catalytic reaction in the confined nanohole 124 are presented in Figure 11. An origami tile 100 nm × 100 nm was engineered to include a surface patch consisting of the two duplex trans-azobenzene locks composed of the strands L 3 /L 3 ′ and L 4 /L 4 ′ associated with the origami raft and patch, respectively, and eight "hinges" linking the patch to the raft (counter to the locking edge). In addition, two handles H a /H b on opposite sides of the patch link the patch to the origami raft ( Figure 11A). Illumination of the origami tile, λ = 365 nm, in the presence of the hairpins H 3 and H 4 , results in the photoisomerization of the trans-azobenzene units to the cis-state, to the separation of the L 3 /L 3 ′ and L 4 /L 4 ′ locks. The concomitant binding of the hairpins H 3 /H 4 to the handles H a /H b bridging the patch to the raft, assist with the opening of the nanohole on the origami raft by turning the patch across the hinges and its stretching over the raft by linking the opened hairpin through hybridization of H a /H 3 and H b /H 4 to the foothold anchoring sites associated with the origami raft. This mechanical, light-induced opening of the patch yields a nanohole (ca. 50 nm diameter) in the origami raft ( Figure 11B). By the reverse photoisomerization of the cisazobenzene units to the trans-azobenzene state, and applying the strands H 3a ′, H 3b ′, H 4a ′, and H 4b ′ as antifuel strands that disconnect the stretching units H 3 and H 4 from the anchoring footholds and the handles, the parent locked origami device is formed. By the reversible and cyclic light-induced unlocking of the trans-azobenzene locks to the cis-state and applying the fuel hairpins H 3 /H 4 , the nanoholes were reopened, and by the reverse light-induced isomerization of the photoisomerizable units to the trans-state and applying the antifuel strands H 3a ′, H 3b ′, H 4a ′, and H 4b ′, the locked origami structure was regenerated. That is, the cyclic photoisomerization of the cisstate origami structure in the presence of the hairpins H 3 , H 4 and the reverse photoisomerization of the trans-azobenzene units, in the presence of H 3a ′, H 3b ′, and H 4a ′, H 4b ′, leads to the dynamic switchable and reversible opening and closing of the nanoholes in the origami rafts ( Figure 11C). The further functionalization of the origami raft allows the switchable "ON" and "OFF" operation of the catalytic hemin/Gquadruplex DNAzyme in the nanometer-sized nanoholes, acting as a confined reaction volume for the switchable catalytic process ( Figure 11D). Two pairs of guanosine-rich nucleic acid strands, with encoded sequences to act a Gquadruplex subunits, G 1x /G 2, were hybridized with appropriate protruding tethers T M1 , T M2 , T M3 , and T M4 , linked as protruding tethers to the upper surface and counter surface of the origami raft at edges ( Figure 11D, inset I). The lightinduced unlocking of the nanoholes, in the presence of K + -ion and hemin, results in the self-assembly of a two K + -ionstabilized G-quadruplex DNAzyme units catalyzing the oxidation of Amplex Red by H 2 O 2 to the Resorufin fluorescent product ( Figure 11D, inset II). The reverse photoinduced closing of the nanohole, in the presence of 18-crown-6-ether (CE), that separates the G-quadruplexes, results in the catalytic Bioconjugate Chemistry pubs.acs.org/bc Review units and the locking of the nanoholes. By the cyclic and reversible light-induced dynamic opening and closing of the nanoholes, in the presence of coadded K + -ions/CE, the catalytic process in the nanoholes is switched between "ON"/"OFF" states ( Figure 11E). Similar "mechanical" dynamic unlocking of nanoholes in origami rafts and operation of cyclic catalytic reactions in the nanoholes were reported using switchable aptamer-ligand complexes as locks. 125 The dynamic equilibration of constitutional dynamic networks (CDNs) and the triggered operation of dissipative out-of-equilibrium systems generating transient chemical intermediates were integrated as functional motives to engineer dissipative self-assembled networks revealing dynamic catalytic properties 126 ( Figure 12A). The system consists of a constitutional dynamic network, CDN T, two coadded duplexes L 1 /T 1 and L 2 /T 2 , and the nicking enzyme, Nt.BbvCI. Triggering CDN T with the fuel strand L 1 ′ displaces the duplex L 1 /T 1 while generating the duplex L 1 /L 1 ′ and the free strand T 1 that hybridizes with the arms associated with constituent BA′. This results in the dynamic transition of CDN T to CDN Q where the constituent BA′ is upregulated. The component B and component T 1 in the systems are pre-engineered, however, to include functional tethers f and e which upon the stabilization of constituent BA′ self-assemble into Mg 2+ -iondependent DNAzyme cleaving F 1 (FAM)/Q 1 (BHQ1)modified substrate (panel III). The L 1 ′ triggered dynamic reconfiguration of CDN T to Q yields the duplex L 1 /L 1 ′ that includes a pre-engineered nicking site being cleaved by Nt.BbvCI, resulting in the release of L 1 that displaces T 1 from constituent BA′, resulting in the transient recovery of CDN Q to CDN T, while separating the Mg 2+ -ion-dependent DNAzyme, associated with BA′. That is, the triggered dynamic reconfiguration of CDN T to CDN Q leads to the transient catalytic operation of the Mg 2+ -ion-dependent DNAzyme which is followed by the fluorescence of the fragment product of the DNAzyme substrate ( Figure 12B). Alternatively, treatment of CDN T with the fuel trigger L 2 ′ stimulates the displacement of the duplex L 2 /T 2 to yield L 2 /L 2 ′ and the released strand T 2 that binds to the "arm" of constituent AA′, leading to the dynamic reconfiguration of CDN T to CDN R, where constituents AA′ and BB′ are upregulated and constituents AB′ and BA′ are downregulated. The component A of constituents AA′ and T 2 is pre-engineered to include the tethers g and h that correspond to G-quadruplex subunit sequences. Thus, the transition of CDN T to CDN R, in the presence of K + -ions and hemin, is accompanied by dynamic formation of the hemin/G-quadruplex DNAzyme, being a part of constituent AA′. The resulting hemin/G-quadruplex DNAzyme catalyzes the oxidation of Amplex Red by H 2 O 2 to form the fluorescent Resorufin that probes the formation of the hemin/G-quadraplex-functionalized constituent AA′. The fuel-triggered duplex L 2 /L 2 ′ inducing the transition of CDN T to CDN R is engineered, however, to include in strand L 2 ′ a nicking site that is cleaved by Nt.BbvCI. Cleavage of L 2 ′ separates the duplex L 2 ′/L 2 , and the released L 2 displaces T 2 from constituent T 2 /AA′ leading to the recovery of CDN R to Bioconjugate Chemistry pubs.acs.org/bc Review CDN T and to the separation of the hemin/G-quadruplex DNAzyme unit associated with constituent AA′ of CDN R. Thus, the L 2 ′-triggered reconfiguration of CDN T to CDN R, in the presence of the duplex L 2 /T 2 and nickase, leads to the dynamic transient operation of the hemin/G-quadruplex DNAzyme ( Figure 12C).

CONCLUSIONS
This Review has addressed an important topic in the rapidly developing area of Systems Chemistry by discussing dynamic catalytic systems driven by nucleic acid−protein (enzyme) conjugates and supramolecular nanostructure composed of nucleic acid scaffolds, coupled to proteins or DNAzymes. These included constitutional dynamic networks (CDNs) that guide biocatalytic cascades and feedback-driven internetwork communicated biocatalytic cascades. In addition, nucleic acid/ enzyme reaction modules guiding dynamic out-of-equilibrium, transient biocatalytic cascades were demonstrated. Alternatively, the triggered switchable mechanical dynamic reconfiguration features of DNA nanostructures were conjugated to the enzyme or DNAzyme functionalities to yield switchable mechanically driven catalytic processes. This has been introduced by highlighting the design of interlocked supramolecular DNA catenane structure that reconfigures, in the presence of appropriate triggers, into switchable DNAzyme structures and by tailoring a supramolecular DNA tweezers structure conjugated to enzyme/DNAzyme constituents. The triggered dynamic opening and closing of the tweezers scaffold led to the switchable operation of a biocatalytic cascade. Also, origami tiles provided functional frameworks for positioning functional units for dynamic catalysis. This has been introduced with the photochemically "mechanical" activation of a NAD + -cofactor-mediated biocatalytic cascade, the rotational, triggered operation of biocatalytic cascade, and the light-induced reversible mechanical unlocking of a nanohole in the origami raft and the use of the nanohole as a confined environment for the switchable operation of a DNAzyme. While substantial progress in developing DNA-based networks and supramolecular DNA structures for operating dynamic catalytic transformations was achieved, interesting and challenging goals are ahead of us. The design of DNAbased constitutional dynamic networks or dissipative, transient nucleic acid-based networks guiding catalytic processes of enhanced complexities and functionalities is desirable. For example, the design of networks guiding artificial photosynthetic and photocatalytic transformations or dynamically driven transcription processes inducing network-guided selective translation of proteins can be envisaged. Similarly, enhancing the complexity of dissipative nucleic acid-based reaction modules to yield gated or cascaded transient machinery is anticipated to establish dynamic, transient transcription of mRNAs and their translation to target proteins, thus emulating biological gene regulation and transcription. Indeed, recent studies demonstrated that dynamically modulated transcription and protein translation are achievable goals. Nonetheless, in the present study, the dynamically triggered catalytic networks operate in homogeneous, in vitro environments. Their integration in cell-like containments, protocells, 127 is certainly a future challenge. The advances in developing artificial protocell assemblies, such as liposomes, 128,129 microdroplets, 130 dendrosomes, 131 polymersomes, 132 and hydrogel microcapsules, 133 provide exciting opportunities to design artificial cells.
Beyond the significant basic and fundamental issues related to dynamic catalytic networks and structures, the identification of practical applications of these concepts is most important. In fact, substantial research efforts were directed to the development of stimuli-responsive materials and their applications for sensing, imaging, controlled release, and switchable material properties. Integrating the elements of dynamic control over stimuli-responsive functions of materials is anticipated to yield temporally modulated, signal-triggered, materials for diverse analytical (sensing and imaging) and medical applications, such as controlled drug release or sense-and-treat systems. Indeed, the control over three stiffness states of a DNA hydrogel matrix by integrated constitutional dynamic network has been recently exemplified. 80 Coupling of catalysts to such systems is anticipated to yield dynamic and transient control over material properties.